Vascular and neural dysfunction in Zucker diabetic fatty rats: a difficult condition to reverse.

AIM: We had previously demonstrated that vascular and neural dysfunction in Zucker diabetic fatty (ZDF) rats is progressive. In this study, we sought to determine whether monotherapy of ZDF rats can reverse the vascular and nerve defects. METHODS: ZDF rats at 16 weeks of age were treated for 12 weeks with the angiotensin-converting enzyme inhibitor enalapril, the antioxidant alpha-lipoic acid, the HMG-CoA reductase inhibitor rosuvastatin or the PPARgamma agonist rosiglitazone. Vasodilation of epineurial arterioles was measured by videomicroscopy. Endoneurial blood flow (EBF) was measured by hydrogen clearance, and nerve conduction velocity was measured following electrical stimulation of motor or sensory nerves. RESULTS: Motor nerve conduction velocity (MNCV), sensory nerve conduction velocity (SNCV) (70 and 77% of control, respectively), EBF (64% of control), and vascular relaxation in response to acetylcholine (50% of control) and calcitonin gene-related peptide (CGRP; 73% of control) are impaired in ZDF rats at 28 weeks of age compared with lean littermate controls. Treatment with enalapril and alpha-lipoic acid attenuated the decrease in MNCV and SNCV. Enalapril, alpha-lipoic acid and rosiglitazone treatment of ZDF rats were partially effective in improving endothelium-dependent vascular dysfunction as measured by vascular relaxation in response to acetylcholine. The same drugs also attenuated the decrease in EBF. However, impairment in vascular relaxation in response to CGRP was improved with only alpha-lipoic acid or rosuvastatin treatment. The increase in superoxide and nitrotyrosine levels in vascular tissue was attenuated by all treatments. CONCLUSIONS: The efficacy of monotherapy treatment of ZDF rats using different classes of drugs for vascular and neural dysfunction once complications have developed did not achieve expected levels. This could be because of the complex aetiology of vascular and neural disease in type 2 diabetes.

Mechanisms of inducible nitric oxide synthase-mediated vascular dysfunction.

OBJECTIVE: Inducible nitric oxide synthase (iNOS) is expressed in arteries during inflammation and may contribute to vascular dysfunction. Effects of gene transfer of iNOS to carotid arteries were examined in vitro in the absence of systemic inflammation to allow examination of mechanisms by which iNOS impairs contraction and relaxation. METHODS AND RESULTS: After gene transfer of iNOS with an adenovirus (AdiNOS), constrictor responses to phenylephrine (PE) and U46619 were impaired. After AdiNOS, inhibition of soluble guanylate cyclase (sGC) with 1H-[1,2,4]oxadiazolo-[4,3,2]quinoxalin-1-one (ODQ) reduced the EC50 for PE from 4.33+/-0.78 micromol/L to 1.15+/-0.43 micromol/L (mean+/-SEM). These results imply that iNOS impairs contraction by activation of the NO/cGMP pathway. Relaxation to acetylcholine (ACh) also was impaired after AdiNOS. Sepiapterin (300 micromol/L), the precursor for tetrahydrobiopterin (BH4), improved relaxation to Ach. Because BH4 is an essential cofactor for production of NO by both iNOS and endothelial nitric oxide synthase (eNOS), these results suggest that iNOS may reduce production of NO by eNOS by limiting availability of BH4. Next, we examined effects of expression of iNOS in endothelium and adventitia. Selective expression of iNOS in endothelium, but not adventitia, impaired contraction to phenylephrine and relaxation to acetylcholine. CONCLUSIONS: We conclude that: (1) iNOS may impair contraction in part by activation of sGC; (2) iNOS impairs relaxation, at least in part, by limiting availability of BH4; and (3) expression of iNOS in endothelium may be a more important mediator of vascular dysfunction than expression of iNOS in adventitia.

Effect of fidarestat and alpha-lipoic acid on diabetes-induced epineurial arteriole vascular dysfunction.

In the present study, the authors examined whether treating streptozotocin-induced diabetic rats with the combination of alpha-lipoic acid and fidarestat, an aldose reductase inhibitor, can promote the formation of dihydrolipoic acid in diabetic animals and thereby enhance the efficacy of alpha-lipoic acid as monotherapy toward preventing diabetic vascular and neural dysfunction. Treating diabetic rats with the combination of 0.25% alpha-lipoic acid (in the diet) and fidarestat (3 mg/kg body weight) prevented the diabetes-induced slowing of motor nerve conduction velocity and endoneurial blood flow. This therapy also significantly improved acetylcholine-mediated vasodilation in epineurial arterioles of the sciatic nerve compared to nontreated diabetic rats. Treating diabetic rats with 0.25% alpha-lipoic acid and fidarestat (3 mg/kg body weight) was equally or more effective in preventing vascular and neural dysfunction than was monotherapy of diabetic rats with higher doses of alpha-lipoic acid or fidarestat. Treating diabetic rats with the combination of 0.25% alpha-lipoic acid and fidarestat (3 mg/kg body weight) significantly improved several markers of oxidative stress and increased the serum levels of both alpha-lipoic acid and dihydrolipoic acid. These studies suggest that combination therapy consisting of alpha-lipoic acid and fidarestat may be more efficacious in preventing diabetes-induced vascular and neural dysfunction in peripheral tissue compared to monotherapy, which requires higher doses to be equally effective. The effect of this combination therapy may in part be due to the increased production and/or level of dihydrolipoic acid.

Gene transfer of inducible nitric oxide synthase impairs relaxation in human and rabbit cerebral arteries.

BACKGROUND AND PURPOSE: These studies evaluated whether gene transfer of inducible nitric oxide synthase (iNOS) is a sufficient stimulus to produce vascular dysfunction in cerebral arteries. METHODS: Intracranial (pial) arteries were dissected from human brain tissue obtained during elective surgery. Isolated human arteries were incubated in vitro with adenovirus containing iNOS (AdiNOS) or a nonexpressive transgene (control, AdBglII) (500 micro L, 3x10(9) plaque-forming units per milliliter), and vascular function was examined 24 hours later. In anesthetized rabbits, AdiNOS or AdBglII (300 microL 1x10(10)) was injected into the cisterna magna. Three days later, the basilar artery was removed, and reactivity was examined ex vivo. RESULTS: In submaximally precontracted vessels, we observed impairment of NO-dependent relaxation in human cerebral arteries after gene transfer of iNOS. Maximum relaxation to bradykinin (1 micromol/L, an endothelium-dependent agonist) was 77+/-11% (mean+/-SE) after AdBglII and 31+/-22% (P<0.05) after AdiNOS. After AdiNOS, responses to nitroprusside (an endothelium-independent NO donor) also were impaired. Responses to both nitroprusside and bradykinin were improved by aminoguanidine (300 micromol/L), an inhibitor of iNOS. AdiNOS produced no change in vasoconstrictor responses to U46619. In basilar arteries from rabbits examined in vitro after gene transfer in vivo, responses to histamine, serotonin, and nitroprusside all were similar after AdiNOS or AdBglII. In contrast, relaxation to acetylcholine was significantly depressed after AdiNOS. Maximum relaxation to acetylcholine (10 micromol/L) was 90+/-3% after AdBglII and 68+/-5% (P<0.05) after AdiNOS. Relaxation of arteries after AdiNOS was improved by aminoguanidine. CONCLUSIONS: These studies suggest that expression of iNOS may impair NO-dependent relaxation in both human and rabbit cerebral arteries.

Effect of M40403 treatment of diabetic rats on endoneurial blood flow, motor nerve conduction velocity and vascular function of epineurial arterioles of the sciatic nerve.

1. To further explore the effect of antioxidants in preventing diabetes-induced vascular and neural dysfunction we treated streptozotocin-induced diabetic rats daily with subcutaneous injections of 10 mg kg(-1) of M40403 (n=11) and compared the results obtained from 17 control rats and 14 untreated diabetic rats. M40403 is a manganese(II) complex with a bis(cyclo-hexylpyridine)-substituted macrocyclic ligand that was designed to be a selective functional mimetic of superoxide dismutase. Thus, M40403 provides a useful tool to evaluate the roles of superoxide in disease states. 2. Treatment with M40403 significantly improved diabetes-induced decrease in endoneurial blood flow, acetylcholine-mediated vascular relaxation in arterioles that provide circulation to the region of the sciatic nerve, and motor nerve conduction velocity (P<0.05). M40403 treatment also reduced the appearance of superoxide in the aorta and epineurial vessels and peroxynitrite in epineurial vessels. Treating diabetic rats with M40403 reduced the diabetes-induced increase in thiobarbituric acid reactive substances in serum but did not prevent the decrease in lens glutathione level. Treating diabetic rats with M40403 did not improve sciatic nerve Na(+)/K(+) ATPase activity or the sorbitol, fructose or myo-inositol content of the sciatic nerve. 3. These studies provide additional evidence that diabetes-induced oxidative stress and the generation of superoxide and perhaps peroxynitrite may be partially responsible for the development of diabetic vascular and neural complications.

NO-dependent vasorelaxation is impaired after gene transfer of inducible NO-synthase.

Proinflammatory stimuli produce expression of inducible NO-synthase (iNOS) within blood vessels and are associated with impaired endothelium-dependent relaxation. Gene transfer of iNOS was used to test the hypothesis that expression of iNOS in blood vessels produces impairment of NO-dependent relaxation as well as contraction. An adenoviral vector containing cDNA for murine iNOS, AdCMViNOS, and a control virus, AdCMVBglII, were used for gene transfer to rabbit carotid arteries in vitro and in vivo. After gene transfer of iNOS in vitro, contractile responses to KCl, phenylephrine, and U46619 were impaired. Relaxation in response to acetylcholine, ADP, A23187, and nitroprusside was also impaired. For example, maximum relaxation of vessels to acetylcholine (10 micromol/L) was 78+/-4% (mean+/-SE) after AdBglII (10(10.5) plaque-forming units) and 34+/-5% after AdiNOS (10(10.5) plaque-forming units, P<0.05). NO-independent relaxation in response to 8-bromo-cGMP and papaverine was not impaired after AdiNOS. Contraction and relaxation were improved in carotid arteries expressing iNOS by aminoguanidine and L-N-iminoethyl lysine, inhibitors of iNOS. After intraluminal gene transfer of iNOS in vivo, contraction of vessels in vitro was normal, but responses to acetylcholine were impaired. In summary, the major finding is that NO-dependent relaxation is impaired in arteries after gene transfer of iNOS in vitro and in vivo. Thus, expression of iNOS per se impairs NO-dependent relaxation.

Effect of antioxidant treatment of streptozotocin-induced diabetic rats on endoneurial blood flow, motor nerve conduction velocity, and vascular reactivity of epineurial arterioles of the sciatic nerve.

We have shown that diabetes-induced reduction in endoneurial blood flow (EBF) and impaired endothelium-dependent vascular relaxation precede slowing of motor nerve conduction velocity (MNCV) and decreased sciatic nerve Na(+)/K(+) ATPase activity. Furthermore, vascular dysfunction was accompanied by an accumulation of superoxide in arterioles that provide circulation to the sciatic nerve. In the present study, we examined the effect that treatment of streptozotocin-induced diabetic rats with antioxidants has on vascular and neural function. Diabetic rats were treated with 0.5% alpha-lipoic acid as a diet supplement or with hydroxyethyl starch deferoxamine (HES-DFO) by weekly intravenous injections at a dose of 75 mg/kg. The treatments significantly improved diabetes-induced decrease in EBF, acetylcholine-mediated vascular relaxation in arterioles that provide circulation to the region of the sciatic nerve, and MNCV. The treatments also reduced the production of superoxide by the aorta and superoxide and peroxynitrite by arterioles that provide circulation to the region of the sciatic nerve. Treating diabetic rats with alpha-lipoic acid prevented the diabetes-induced increase in thiobarbituric acid-reactive substances in serum and significantly improved lens glutathione levels. In contrast, treating diabetic rats with HES-DFO did not prevent diabetes-induced changes of either of these markers of oxidative stress. Diabetes-induced increase in sciatic nerve conjugated diene levels was not improved by treatment with either alpha-lipoic acid or HES-DFO. Treating diabetic rats with alpha-lipoic acid but not HES-DFO partially improved sciatic nerve Na(+)/K(+) ATPase activity and myo-inositol content. The increase in sciatic nerve sorbitol levels in diabetic rats was unchanged by either treatment. These studies suggest that diabetes-induced oxidative stress and the generation of superoxide may be partially responsible for the development of diabetic vascular and neural complications.

Arachidonate dilates basilar artery by lipoxygenase-dependent mechanism and activation of K(+) channels.

Dilatation of cerebral arterioles in response to arachidonic acid is dependent on activity of cyclooxygenase. In this study, we examined mechanisms that mediate dilatation of the basilar artery in response to arachidonate. Diameter of the basilar artery (baseline diameter = 216 +/- 7 micrometer) (means +/- SE) was measured using a cranial window in anesthetized rats. Arachidonic acid (10 and 100 microM) produced concentration-dependent vasodilatation that was not inhibited by indomethacin (10 mg/kg iv) or N(G)-nitro-L-arginine (100 microM) but was inhibited markedly by baicalein (10 micrometerM) or nordihydroguaiaretic acid (NDGA; 10 microM), inhibitors of the lipoxygenase pathway. Dilatation of the basilar artery was also inhibited markedly by tetraethylammonium ion (TEA; 1 mM) or iberiotoxin (50 nM), inhibitors of calcium-dependent potassium channels. For example, 10 microM arachidonate dilated the basilar artery by 19 +/- 7 and 1 +/- 1% in the absence and presence of iberiotoxin, respectively. Measurements of membrane potential indicated that arachidonate produced hyperpolarization of the basilar artery that was blocked completely by TEA. Incubation with [(3)H]arachidonic acid followed by reverse-phase and chiral HPLC indicated that the basilar artery produces relatively small quantities of prostanoids but large quantities of 12(S)-hydroxyeicosatetraenoic acid (12-S-HETE), a lipoxygenase product. Moreover, the production of 12-HETE was inhibited by baicalein or NDGA. These findings suggest that dilatation of the basilar artery in response to arachidonate is mediated by a product(s) of the lipoxygenase pathway, with activation of calcium-dependent potassium channels and hyperpolarization of vascular muscle.

Gene transfer of endothelial nitric oxide synthase improves relaxation of carotid arteries from diabetic rabbits.

BACKGROUND: Diabetes mellitus is associated with impairment of NO-mediated vascular relaxation. The purpose of this study was to determine whether adenovirus-mediated gene transfer of endothelial NO synthase (eNOS) or Cu/Zn superoxide dismutase (SOD1) improves responsiveness to acetylcholine in alloxan-induced diabetic rabbits. METHODS AND RESULTS: After 8 weeks, plasma glucose was greater in diabetic rabbits (418+/-35 mg/dL) (mean+/-SEM) than in normal rabbits (105+/-4 mg/dL). Carotid arteries were removed and cut into ring segments. Arteries were incubated for 2 hours with adenoviral vectors driven by a CMV promoter expressing beta-galactosidase (beta-gal), eNOS, SOD1, or vehicle. After incubation with virus, arteries were incubated for an additional 24 hours to allow transgene expression. Vascular reactivity was examined by recording isometric tension. After precontraction with phenylephrine, responses to the endothelium-independent vasodilator sodium nitroprusside were similar in diabetic and normal arteries. Endothelium-dependent relaxation to acetylcholine (3x10(-6) mol/L) was significantly less in arteries from diabetic animals (68+/-5%) than in normal vessels (90+/-3%). Adenoviral transfection of arteries with eNOS improved relaxation in response to acetylcholine in diabetic (EC(50) eNOS=0.64+/-0.12x10(-7) mol/L versus vehicle =1. 70+/-0.43x10(-7) mol/L) but not normal arteries. Vasorelaxation in response to acetylcholine was inhibited by N(omega)-nitro-L-arginine (100 micromol/L) in all groups. Responses to acetylcholine were unchanged after gene transfection of SOD1 or beta-gal in arteries from diabetic or normal rabbits. CONCLUSIONS: Adenovirus-mediated gene transfer of eNOS, but not SOD, improves impaired NO-mediated relaxation in vessels from diabetic rabbits.

Slowing of motor nerve conduction velocity in streptozotocin-induced diabetic rats is preceded by impaired vasodilation in arterioles that overlie the sciatic nerve.

Diabetes mellitus produces marked abnormalities in motor nerve conduction, but the mechanism is not clear. In the present study we hypothesized that in the streptozotocin (STZ)-induced diabetic rat impaired vasodilator function in arterioles that provide circulation to the region of the sciatic nerve is associated with reduced endoneural blood flow (EBF) and that these defects precede slowing of motor nerve conduction velocity, and thereby may contribute to nerve dysfunction. As early as three days after the induction of diabetes endoneural blood flow was reduced in the STZ-induced diabetic rat. Furthermore, after 1 week of diabetes acetylcholine-induced vasodilation was found to be impaired. This was accompanied by an increase in the superoxide level in arterioles that provide circulation to the region of the sciatic nerve as well as changes in the level of other markers of oxidative stress including an increase in serum levels of thiobarbituric acid reactive substances and a decrease in lens glutathione level. In contrast to the vascular related changes that occur within 1 week of diabetes, motor nerve conduction velocity and sciatic nerve Na+/K+ ATPase activity were significantly reduced following 2 and 4 weeks of diabetes, respectively. These studies demonstrate that changes in vascular function in the STZ-induced diabetic rat precede the slowing of motor nerve conduction velocity (MNCV) and are accompanied by an increase in superoxide levels in arterioles that provide circulation to the region of the sciatic nerve.

Adenovirus-mediated gene transfer is augmented in basilar and carotid arteries of heritable hyperlipidemic rabbits.

BACKGROUND AND PURPOSE: There are major differences in susceptibility of intracranial and extracranial arteries to atherosclerosis. The goal of this study was to examine adenovirus-mediated gene transfer to basilar and carotid arteries of Watanabe heritable hyperlipidemic (WHHL) rabbits, which have spontaneous hypercholesterolemia and atherosclerosis, and normal New Zealand White (NZW) rabbits. We used 2 different adenoviral vectors, driven by either cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoters. METHODS: Basilar and carotid arteries were removed from WHHL and NZW rabbits and cut into rings. The arteries were incubated with an adenoviral vector that expresses beta-galactosidase and is driven by either a cytomegalovirus (CMV) or Rous sarcoma virus (RSV) promoter (AdCMVbetagal or AdRSVbetagal). Arteries were incubated with virus for 2 hours, and then incubated in medium for 24 hours to allow expression of transgene. Transgene expression was assessed by enzyme activity (Galacto-Light assay) and by a histochemical method after X-Gal staining. RESULTS: After gene transfer, beta-galactosidase was expressed in endothelium and adventitia but not media. There were moderately severe atherosclerotic lesions in carotid arteries and early lesions in basilar arteries. Enzyme activity after gene transfer with AdCMVbetagal (3x10(11) particles/mL) was greater in the basilar artery of WHHL than NZW (137+/-40 versus 25+/-10 mU/mg protein, P<0.05) (mean+/-SE) and in the carotid artery (133+/-27 versus 34+/-11 mU/mg protein, P<0.05). After gene transfer with AdRSVbetagal, transgene expression was similar in arteries from WHHL and normal NZW rabbits. CONCLUSIONS: This is the first study to examine gene transfer to intracranial and extracranial arteries from atherosclerotic animals. The findings suggest that an adenoviral vector with a CMV, but not RSV, promoter provides greater transgene expression in the basilar and carotid arteries from spontaneously atherosclerotic rabbits than from normal rabbits.

Effects of beta-blockade on neurohumoral responses and neurochemical markers in pacing-induced heart failure.

We investigated neurohumoral profiles and transmitter and neuroenzyme markers of cardiac autonomic innervation in control (unpaced) dogs and three groups of dogs with pacing-induced heart failure (paced, paced + beta-adrenergic blockade, and paced + cardiac denervation). Left ventricular ejection fraction decreased significantly and to a comparable extent in all paced groups. Pacing increased plasma norepinephrine (NE); increases in NE were not attenuated but instead tended to be exaggerated by treatment with propranolol or cardiac denervation. Atrial hypertrophy occurred in all paced groups compared with the control group. However, atrial and right ventricular hypertrophy were not as pronounced in the paced plus cardiac denervation group as in the paced and paced plus propranolol groups. Pacing also depleted neuropeptide Y and NE from all heart chambers; propranolol treatment did not modify these local tissue changes. Pacing caused selective depletion of neuroenzymes predominantly in the left ventricle; again, propranolol did little to modify these changes. In this study of paced animals with experimentally maintained cardiac dysfunction, failure to modify noradrenergic responses with intrapericardial cardiac denervation suggests that noncardiac sources contribute predominantly to high plasma NE. Failure to modify neurohumoral, neuropeptide, and neuroenzyme responses with beta-antagonist suggests this treatment has little practical direct influence on sympathetic vasomotor activity or neuronal function in heart failure.

Immunolocalization of GLUT-1 glucose transporter in rat skeletal muscle and in normal and hypoxic cardiac tissue.

We compared the expression and cell-type localization of GLUT-1 mRNA and protein between cardiac and skeletal muscle of normal rats. Also, since we recently showed that cardiac GLUT-1 is upregulated in rats exposed to hypobaric hypoxia, we examined the cellular localization of GLUT-1 in cardiac tissue of normal and hypoxic rats. Confocal light microscopy and double immunofluorescent labeling revealed intense localization of GLUT-1 around neurofilament immunoreactivity within gastrocnemius muscle consistent with the previously described localization of large amounts of GLUT-1 in perineurial sheaths of skeletal muscle. However, using the same methods, we were unable to visualize GLUT-1 adjacent to nerve fibers in numerous sections of right or left ventricles or atria. Compared with skeletal myoctes, however, GLUT-1 immunofluorescence among cardiomyocytes was much more intense, particularly along the plasma membrane and especially intercalated discs. GLUT-1 immunofluorescence was also seen within the walls of arterioles within the heart. The predominant localization of GLUT-1 expression to cardiomyocytes in heart tissue was confirmed by in situ mRNA hybridization to digoxigenin-conjugated GLUT-1 cDNA. Northern blot analysis demonstrated that GLUT-1 mRNA was increased severalfold in the cardiac tissues compared with skeletal muscle. Although we detected GLUT-1 protein by immunoblotting of detergent extracts of the heart, we could not detect GLUT-1 in similar extracts of skeletal muscle. The cell type distribution of GLUT-1 in hearts of hypoxic rats was not different by immunohistochemistry from normals. These data indicate that 1) the cell-type distribution of GLUT-1 in the heart differs markedly from that in skeletal muscle. GLUT-1 in cardiac tissue, unlike skeletal muscle, is predominantly expressed within myocytes. 2) Cardiac GLUT-1 is not located along nerve fibers. 3) GLUT-1 mRNA and protein levels in cardiac tissue are considerably greater than in skeletal muscle. 4) The hypoxia-induced increase in cardiac GLUT-1 that we previously reported must occur within cardiomyocytes.

The role of arginine vasopressin on peripheral cardiac parasympathetic nerve function in the rat.

In rats, arginine vasopressin augments bradycardia associated with baroreflex activation. We investigated whether modulation of peripheral cardiac parasympathetic nerve function by AVP may play a role in this effect. To accomplish this we utilized an in vivo model with which we previously demonstrated both adrenergic and peptidergic modulation of cardiac parasympathetic nerve function. Urethane-anesthetized rats (250-350 g) were prepared with arterial and venous catheters and ECG leads. The cervical vagi were sectioned, and propranolol (1 mg/kg, i.v.) was administered to eliminate reflex changes in heart rate. To investigate potential preganglionic modulation by AVP, the right vagus nerve was electrically stimulated (0.5 mA; 0.5 msec; 1-10 Hz). To observe postganglionic effects through nicotinic activation, carbachol (a mixed nicotinic and muscarinic agonist) was injected (0.5 to 4.0 micrograms/kg, i.v.). To observe direct cholinergic effects at the SA node, methacholine (a pure muscarinic agonist) was injected (0.5 to 4.0 micrograms/kg). All three trials were performed before (control) and during AVP infusion (20 micrograms.kg.min). No consistent, significant differences in vagal-, carbachol- or methacholine-induced bradycardia were observed between control and AVP groups. Since endogenous plasma levels of AVP in the control situation may have saturated any vasopressinergic effect prior to AVP infusion, the experiments were repeated in Brattleboro rats, genetically deficient in AVP. Again, no consistent differences in heart rate responses to parasympathetic activation were noted between control and AVP-infused groups. These results suggest that in rats, vasopressinergic augmentation of baroreflex-induced bradycardia is not mediated by an effect on the peripheral cardiac parasympathetic innervation. However, it remains to be investigated whether AVP-mediated sympathetic withdrawal disinhibits cardiac parasympathetic nerve function.

Transient depression of responses to sympathetic nerve stimulation overlying a subendocardial infarct.

In previous work, the normal epicardial rim overlying a subendocardial infarct was demonstrated to be parasympathetically denervated. In the present study, we determined responses of effective refractory period (ERP) in this rim during sympathetic nerve stimulation (SNS). Eighteen dogs were studied 1-3 days after a 1-h or permanent coronary artery occlusion (group I). SNS shortened ERP in sites basal, septal, and lateral in the rim by 8 +/- 2, 7 +/- 2, and 7 +/- 2% (SE), respectively, which were similar to sites remote from the infarct (10 +/- 1%). These results were not altered by site of infarction or by atropine administration. To eliminate dissection of the coronary vessel and spontaneous ventricular tachycardia, 19 dogs were studied 6 h after a permanent bead embolization of a coronary artery (group II). In contrast to group I, ERP shortening in the rim sites of group II was depressed (3 +/- 3, 0 +/- 2, and 1 +/- 2%, respectively) compared with remote sites (10 +/- 1%, P < 0.05). In this group, collateral blood flow in the rim was no different than remote epicardium before and during SNS, and norepinephrine shortened ERP in the rim equivalent to remote sites. In an additional 31 animals (group III), the alteration in ATP-dependent K+ channel function was evaluated. Pretreatment with glyburide (an ATP-dependent K+ channel blocker) preserved ERP response to SNS (9 +/- 1% shortening of ERP vs. 12 +/- 2% at baseline) compared with only 3 +/- 0% shortening of ERP with vehicle (P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Pretranslational regulation of two cardiac glucose transporters in rats exposed to hypobaric hypoxia.

To investigate the mechanism by which cardiac glucose utilization increases during hypoxia and increased work load, we studied the effect of 2 and 14 days of hypobaric hypoxia on the expression of two subtypes of the facilitative D-glucose transporter, the GLUT-4 or "insulin-regulatable" isoform and the GLUT-1 isoform thought to mediate basal transport. Rats lose weight when exposed to hypobaric hypoxia, so fasting controls were used in the 2-day studies and pair-fed controls in the 14-day experiments. Hypobaric hypoxia (PO2 69 mmHg) resulted in right ventricular (RV), but not left ventricular (LV), hypertrophy. RV and LV GLUT-1 mRNA levels increased 2- to 3-fold after 2 days and 1.5- to 2-fold after 14 days of hypobaric hypoxia compared with both fasted rats and normal controls. RV GLUT-1 protein increased approximately 3-fold and LV GLUT-1 protein increased 1.5-fold after 14 days of hypobaric hypoxia vs. both pair-fed and normal controls. RV GLUT-4 mRNA decreased to 26% and RV GLUT-4 protein decreased to 54% of normal control levels as a result of 2 days of hypobaric hypoxia. RV GLUT-4 mRNA decreased to 64% of normal control levels with no change in RV GLUT-4 protein as a result of 2 days of fasting. We conclude that hypobaric hypoxia increases cardiac GLUT-1 expression at the pretranslational level in both ventricles. The greater increase in GLUT-1 protein on the right suggests an additive effect of pressure overload. GLUT-4 expression is reduced early in the development of RV hypertrophy.

Sites at which neuropeptide Y modulates parasympathetic control of heart rate in guinea pigs and rats.

Immunohistological evidence indicates that neuropeptide Y (NPY) is present in the cardiac innervation of numerous species. The present experiments determined if NPY influences in vivo parasympathetic control of heart rate in guinea pigs and rats by either pre- or postganglionic mechanisms or by an interaction at muscarinic receptors at the sino-atrial node. Urethane-anesthetized animals were prepared with arterial and venous catheters, and ECG leads. The cervical vagi were sectioned and propranolol was administered to minimize reflex changes in heart rate. Methacholine injection, carbachol injection, or electrical stimulation of the peripheral end of the vagus nerve was performed to activate the neuroeffector site, intracardiac ganglion cells, or preganglionic neurons, respectively. All three trials were performed before, during, and after NPY infusion. No differences in methacholine- or carbachol-induced bradycardia were observed between control and NPY groups in either species. NPY infusion inhibited vagal-mediated bradycardia in guinea pigs and in rats. However, NPY inhibited vagal-mediated bradycardia at a lower dose in guinea pigs (1 microgram/kg/min) than in rats (4 micrograms/kg/min). These data indicate that NPY modulates cardiac vagal preganglionic, but not postganglionic nerve function or neuroeffector sites at the sino-atrial node, in guinea pigs and rats. Furthermore, due to the different effective dosages, NPY may play a greater modulatory role in guinea pigs than in rats.

Compensatory recovery of parasympathetic control of heart rate after unilateral vagotomy in rabbits.

Compensatory recovery by the intact vagal innervation after unilateral vagotomy was investigated by measuring parasympathetic-mediated control of heart rate in beta-adrenergic-blocked rabbits. Direct contralateral vagal nerve stimulation produced greater bradycardia in anesthetized rabbits with chronic vagotomy compared with acutely vagotomized controls. Vagal stimulation during acetylcholinesterase inhibition by physostigmine and direct neuroeffector stimulation by methacholine indicated that a change in metabolism of the neurotransmitter or an increased sensitivity of the tissue to acetylcholine were not responsible for augmentation of vagal responses. Baroreflex control of heart rate in response to an increase in arterial pressure was also tested in urethan-anesthetized rabbits. There was a significant reduction in the prolongation of the R-R interval during baroreflex activation acutely after midcervical vagotomy. These values were subsequently above control levels in rabbits 28 days after vagotomy. In conscious rabbits, the decrease in baroreflex control of heart rate progressively recovered to control levels within 6 days. These results suggest that the recovery mechanism after unilateral vagotomy may be related to peripheral and central compensatory changes in the intact contralateral vagus nerve.

Alterations in cardiac parasympathetic indices in STZ-induced diabetic rats.

Autonomic neuropathy involving parasympathetic innervation is a complication of diabetes mellitus. Biochemical and morphological indices of the parasympathetic innervation of the heart were investigated in rats after diabetes mellitus was induced with streptozocin (STZ). Choline acetyltransferase (CAT) activity was used as a biochemical marker for parasympathetic innervation. Total CAT activity within the hearts of diabetic rats was unchanged after 1 and 2 wk of diabetes and was significantly reduced after 4, 8, and 12 wk. Morphological changes within the cardiac portion of the parasympathetic innervation were assessed at 8 wk when CAT activity was decreased. In diabetic rats, there was a reduction in both cardiac ganglion cell size and number. In contrast, in insulin-treated STZ-induced diabetic rats, ganglion cells were similar in size and number to those in a control group given 3-O-methylglucose to prevent induction of diabetes mellitus by STZ. Thus, diabetes mellitus is associated with alterations in cardiac parasympathetic innervation in rats, and supplemental insulin protects against these changes. These alterations may contribute to impaired parasympathetic neural control of the heart in diabetes mellitus.

Presynaptic regulation of cardiac sympathetic function in hypoxic guinea pigs.

In the normal heart, presynaptic cholinergic muscarinic and alpha 2-adrenergic mechanisms modify the fractional rate constant for norepinephrine (NE) synthesis (kNE), an index of sympathetic neural function. To evaluate presynaptic regulation of kNE, conscious guinea pigs subjected to normoxia and then hypoxia (n = 7-8 in each group) were pretreated with 1) vehicle; 2) a cholinergic muscarinic antagonist, methyl atropine; 3) an alpha 2-antagonist, yohimbine; or 4) a combination of the two. An increase of kNE was determined from incorporation of radiolabeled tyrosine into NE in a control period (arterial PO2 130 +/- 1.7 Torr, PCO2 36 +/- 0.5 Torr) and during a hypoxic state (PO2 49.6 +/- 1.0 Torr, PCO2 36 +/- 0.5 Torr). Hypoxia activated kNE in the atrioventricular node and right ventricular moderator band in vehicle-treated animals (P less than 0.05). Sympathetic activation was more general, however, because alpha 2-presynaptic influence acted to limit kNE in all tissues tested (P less than 0.05) except muscle, spleen, and posterior left ventricle. Cholinergic muscarinic presynaptic restraint on kNE was detected during hypoxia only in the left atrial appendage and lung (P less than 0.05). These data indicate that hypoxia increases kNE in the heart, but restraint by cholinergic muscarinic and alpha 2-adrenergic presynaptic mechanisms limits increases in neurotransmitter synthesis and noradrenergic activation regionally.